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Abstract

Background

Epidemiological studies on risk factors for colorectal cancer (CRC) have mainly focused
on diet, and being overweight is now recognized to contribute significantly to CRC
risk. Overweight and obesity are defined as an excess of adipose tissue mass and are
associated with disorders in lipid metabolism. Peroxisome proliferator-activated receptors
(PPARs) and retinoid-activated receptors (RARs and RXRs) are important modulators
of lipid metabolism and cellular homeostasis. Alterations in expression and activity
of these ligand-activated transcription factors might be involved in obesity-associated
diseases, which include CRC. Cyclooxygenase-2 (COX-2) also plays a critical role in
lipid metabolism and alterations in COX-2 expression have already been associated
with unfavourable clinical outcomes in epithelial tumors. The objective of this study
is to examine the hypothesis questioning the relationship between alterations in the
expression of nuclear receptors and COX-2 and the weight status among male subjects
with CRC.

Method

The mRNA expression of the different nuclear receptor subtypes and of COX-2 was measured
in 20 resected samples of CRC and paired non-tumor tissues. The association between
expression patterns and weight status defined as a body mass index (BMI) was statistically
analyzed.

Results

No changes were observed in PPARγ mRNA expression while the expression of PPARδ, retinoid-activated
receptors and COX-2 were significantly increased in cancer tissues compared to normal
colon mucosa (P ≤ 0.001). The weight status appeared to be an independent factor, although we detected
an increased level of COX-2 expression in the normal mucosa from overweight patients
(BMI ≥ 25) compared to subjects with healthy BMI (P = 0.002).

Conclusion

Our findings show that alterations in the pattern of nuclear receptor expression observed
in CRC do not appear to be correlated with patient weight status. However, the analysis
of COX-2 expression in normal colon mucosa from subjects with a high BMI suggests
that COX-2 deregulation might be driven by excess weight during the colon carcinogenesis
process.

Background

Approximately 5% of individuals will develop colorectal carcinoma during their lifetime
[1]. This disease typically progresses from adenomatous polyps and dysplastic polyps
to invasive carcinoma [2,3]. CRC seems particularly susceptible to specific nutritional factors and dietary habits
(for review [4]). Indeed, excessive consumption of calories from fat is thought to be largely responsible
for the increasing incidence of CRC in western countries. Moreover, overweight and
obesity status associated with high body mass indices have been correlated with a
higher risk of developing CRC [5,6]. Nevertheless, the mechanisms of why and how excess weight increases cancer risk
are only slowly emerging. One proposed mechanism is the rise of insulin resistance
resulting in hyperinsulinemia that can cause growth-promoting effects [7].

Hyperinsulinemia as well as hyperlipidemia, hypertension, overweight and type II diabetes
are metabolic disorders which might be caused by alterations in the homeostasis of
the metabolism of fatty acids [8]. These obesity-related symptoms could affect the integrity of colon tissue homeostasis
and therefore be involved in CRC etiology [9]. Anti-diabetic treatments initially used for improving parameters such as insulin
sensitivity have been shown to be able to inhibit colon carcinogenesis in rodent models
[10,11] and to promote differentiation of colon cancer cells [12]. Moreover, concomitant suppression of hyperlipidemia and polyp formation were observed
in APC-deficient mice treated with insulin-sensitizing drugs called thiazolidinediones
(TZD) [13]. In such a context, PPAR family members initially recognized for their involvement
in regulating fat metabolism and adipogenesis have emerged as attractive targets for
therapeutic approaches for obesity and CRC. Indeed, PPARγ agonists including anti-diabetic
agents, polyunsaturated fatty acids as well as non-steroidal anti-inflammatory drugs
(NSAID) [14,15] have been demonstrated to affect proliferation and differentiation in cancer cell
lines [16]. Moreover, both anti-proliferative effects of PPARγ observed in vitro [12] and inactivating mutations in the PPARγ gene found in colon tumors [17] provide evidence for a tumor suppressor function. This is also supported by the finding
that an increased risk of polyp occurrence in colon mucosa was found to be significantly
associated with a decrease in PPARγ mRNA expression [18]. Another isotype, PPARδ, may also play an important role in the process of colon
carcinogenesis since it has been efficiently targeted by hypolipidemic and hypoglycemic
drugs [19]. However, PPARδ might display distinct functions in lipid metabolism and colon carcinogenesis.
Indeed and in contrast to PPARγ, PPARδ was found frequently overexpressed in colon
cancer cells [20] and tumors of chemically-initiated animals [21]. Nevertheless, the role of PPARδ in colonic epithelium stays unclear due to conflicting
evidence [22].

The transcriptional activity of PPAR depends on the presence of the retinoic X receptor
(RXR), activated by 9-cis retinoic acid (9-cis RA). Heterodimerization with RXR is
essential for the activity of all class II nuclear receptors [23] and explains how fatty acids and retinoids control lipid metabolism [24]. The active forms of vitamin A, 9-cis RA and all-trans retinoic acid (atRA), also
exhibit anti-tumoral properties in many tissues mainly through RXR and retinoic acid-activated
receptor (RAR) binding. Indeed, retinoids have displayed chemopreventive and chemotherapeutic
activities with regard to their capacity to induce cell differentiation and apoptosis
(for review [25]). RXRα is by far the most prevalent isoform in the colon, while RXRβ and RXRγ are
expressed at low levels [26]. All three RAR isotypes, α, β, and γ, are expressed and induced by retinoids in colon
cancer cell lines [27]. Alterations in retinoid-activated receptor expression and biological activity have
been observed both in vivo and in vitro [28]. However, potential alterations have been poorly investigated in CRC although they
may affect the response of target cells to retinoid and lipid derivatives.

COX-2 is a key enzyme in lipid metabolism and is well-known to convert arachidonic
acid to growth-regulating molecules such as prostaglandins. COX-2, activated by growth
factors and pro-inflammatory cytokines, has been shown to be overexpressed in several
epithelial cancers including CRC [29,30]. This enzyme might mediate the promotion of colon carcinogenesis by metabolic disorders
and inflammation and several lines of evidence indicate that COX-2 might be regulated
by PPARγ [31] and RARβ activation in cancer cells [32,33] although the mechanism is unclear. It was also shown that deregulations in nuclear
receptor expression might promote COX-2 upregulation [34].

In the current report, our interest was (i) to evaluate alterations in the expression
of the different nuclear receptors and COX-2 in several colon cancer specimens from
patients undergoing surgery to remove tumors and (ii) to clarify whether or not the
expression of nuclear receptors and COX-2 was affected by the weight status of patients
with CRC.

Methods

Patients and samples

Tumor samples and normal adjacent tissue microscopically confirmed to be free of cancer
were obtained from 20 patients undergoing surgery for colorectal cancer at the St.
André Hospital (Bordeaux, France) from January 2003 to September 2004. Following surgery,
samples from tumor and adjacent normal mucosa were frozen in liquid nitrogen and stored
at -80°C for subsequent RNA extraction. All included patients were men without neoadjuvant
treatment. Patients suffering from familial cancer syndromes were excluded. Information
concerning age, BMI (weight (kg)/height (m)2), tumor site, pTNM and Dukes stage are indicated in Table 1. BMI was found not to be correlated with height in this population (r = 0.07, n = 20), but strongly correlated with weight (r = 0.89, n = 20). Segregation of patients into BMI groups corresponded to the following
categorizations: 18.5–25 kg/m2 (healthy weight), ≥ 25 kg/m2 (excess weight group including overweight/pre-obese/obese subjects).

Real-time Polymerase Chain Reaction (PCR)

Real-time quantitative PCR involving LightCycler™ technology (Roche Diagnostics, Mannheim,
Germany), was performed according to the protocol recommended by the manufacturer
and previously described [34]. SYBR green I fluorescence dye was sufficiently sensitive to accurately detect amplified
products from all target cDNA (PPARδ, PPARγ, RARα, RXRα and COX-2) except for RARβ
and RARγ amplified product detected using dual-labeled and specific TaqMan fluorogenic
probes. Quantification data were analyzed using the LightCycler Relative Quantification
Software (Roche Diagnostics, Mannheim, Germany). The software provides a crossing
point (Cp), defined as the PCR cycle number, function of the log of the DNA concentration
(in ng). A standard curve is a plot of the Cp versus the amount of initial cDNA used
for amplification. Standard curves were used to estimate the concentration of both
the target and the reference gene in each sample. This software provides a calibrator-normalized
relative quantification including PCR efficiency correction considering then the difference
existing between amplification efficiencies of reference and target cDNAs. cDNA from
tissue samples from patient A was arbitrarily chosen to be the calibrator. The cDNA
calibrator was used in all experiments. Results are expressed as the target:reference
ratio divided by the target:reference ratio of the calibrator. Primers and fluorogenic
probes were purchased from Proligo France (Paris, France). Each probe was synthesized
with the fluorescent reporter dye FAM (6-carboxy-fluorescein) attached to the 5'-end
and a quencher dye TAMRA (6-carboxy-tetramethyl-rhodamine) to the 3'-end. Specificity
of primers was validated through the verification of RT-PCR product specificity. RT-PCR
products were subjected to analysis by electrophoresis on a 1.5% agarose gel and resulted
in a single product with the desired length (β2-microglobulin, 112 bp; PPARδ, 139
bp; PPARγ, 144 bp; RARα, 235 bp; RARβ, 133 bp; RARγ, 167 bp; RXRα, 142 bp; COX-2,
130 bp). The identity of amplified products were assessed by sequencing with a Dye
Terminator Reaction Cycle Kit (Perkin-Elmer, Norwalk, CT) and were analyzed on an
ABI PRISM™ 377 automated DNA sequencer (Perkin-Elmer). The forward and reverse primer
sequences and the probes were as follows:

Statistical methods

Statistical analyses were carried out using the Windows SPSS® 9.0 software package. Associations between clinicopathological variables (age, TNM
and Dukes stage (Dukes B vs. C vs. D), BMI (BMI < 25 vs. BMI ≥ 25), tumor site (descendant/sigmoid colon/rectum vs. ascendant colon)) were assessed by Spearman's correlation coefficient test. Associations
between mRNA expression levels were tested for correlation by Spearman's test and
mRNA levels were compared with regard to clinicopathological features. Specifically,
comparison of mRNA expression levels in healthy tissue with regard to BMI and tumor
site was performed using the Mann-Whitney U test. The Kruskal-Wallis test was used
to assess for significant differences in mRNA expression with regard to Dukes stage
(Dukes B vs. C vs. D). The significance of differences in mRNA expression levels between healthy mucosa
and tumor tissue was evaluated using the Wilcoxon-test. A P value < 0.05 was considered as significant.

Results

Samples of CRC specimens and adjacent non-neoplastic colonic mucosa were collected
from patients undergoing surgery. Pertinent clinical and pathological data are listed
in Table 1. All patients were men with a median age of 72 years old (range 41–87). Six patients
(30%) had Dukes' B tumors, nine patients (45%) were classified as Dukes' C or as Dukes'
D (25%). Eleven patients (55%) had a BMI greater than 25, and were designated as overweight.
Four of these had a BMI value above 30, corresponding to obesity status. No correlation
was found between age, BMI, and tumor classification.

Nuclear receptors and COX-2 were detectable by quantitative real-time RT-PCR in all
normal-looking tissue and tumor samples. The median mRNA expression values are shown
in Table 2. The median relative PPARγ expression level in tumors remained unchanged as compared
to normal mucosa. Indeed, among 20 investigated cancer tissue samples, PPARγ increased
between 1.5- and 4-fold in 35% (n = 7), while we noted a 1.5- to 6-fold decrease in
25% (n = 5). No changes were observed in the remaining 40% (n = 8). In contrast, the
expression of PPARδ in tumors was significantly upregulated (1.54 vs. 1.30, P = 0.001) relative to normal mucosa. All retinoid nuclear receptors were also upregulated
in tumor tissues compared to healthy mucosa (n = 20, P < 0.001). Expression levels were increased by the following percentages: RXRα 26.7%,
RARα 27.4%, RARβ 54.9%, and RARγ 149.6%. COX-2 mRNA expression was multiplied by 8.5
between normal and tumor tissues (P < 0.001). Relationships between nuclear receptor and COX-2 mRNA expression were tested
statistically and listed in Tables 3 and 4 (see also additional file 1). Further combined analysis of receptor and COX-2 mRNA expression levels with regard
to Dukes' stages and tumor localisations did not display any significant statistical
difference. COX-2 expression was not correlated with tumor stages and localisations
(data not shown), but was associated with RARα and RARβ mRNA expression in tumor tissue
(Table 4).

Differences in expression of nuclear receptors and COX-2 between normal and tumor
tissues (Table 2) were also observed when patients were segregated into groups with low and high BMI
(BMI < 25 vs. BMI ≥ 25) (Tables 5 and 6). We also compared the expression of nuclear receptors and COX-2 in healthy mucosa
regarding the BMI of patients (Table 7). Statistics revealed that COX-2 expression is significantly increased in the normal-looking
mucosa from patients with the highest BMI (Table 7).

Discussion

Nuclear receptors are involved in many cellular processes from embryonic development
to cell death. Dysfunction of nuclear receptor signaling can lead to proliferative
and metabolic diseases such as cancer and obesity. In the current report, we assessed
the mRNA expression levels of nuclear receptors and COX-2 in 20 CRC specimens and
sought a possible relationship with patient's weight status defined by BMI ranging
from 18.7 to 38.7.

PPARγ constitutes the most extensively studied of the three PPAR subtypes (α, β, γ)
since its function relates to lipid metabolism as well as cell differentiation, apoptosis
and cancer. PPARγ can be activated by certain lipids and derivatives and by anti-diabetic
agents. Activated PPARγ has been shown able to stimulate differentiation and apoptosis
in cancer cells from various origins [35-38]. Nevertheless, in contrast with results generated in vitro, data concerning PPARγ expression in human cancer specimens raised questions about
the anti-neoplastic activity of the receptor in vivo. For example, PPARγ was found highly expressed in ovarian carcinoma [39] and its overexpression in pancreatic carcinoma was associated with poor prognosis
[40]. By contrast, our data, in agreement with others [41], showed PPARγ expression globally unchanged in CRC compared with adjacent normal
tissues, although Dubois et al. [42] found a marked increase of PPAR mRNA expression in four CRC samples and in different
colon cancer cell lines. Discrepancies might be attributed to germline mutations in
the adenomatous polyposis gene (APC). Indeed, PPARγ has been involved in increasing
resistance towards carcinogens by preventing the accumulation of β-catenin, which
is regulated by APC. However, PPARγ functions are lost when APC is mutated [43]. Another report has shown that deregulated APC/β-catenin indirectly induced aberrant
PPARγ overexpression [44], explaining previous experimental data in APCMin/+ mice showing a promoting effect of PPARγ on carcinogenesis [45]. This has relevance for humans because mutations in the tumor suppressor gene APC are the initiating event in about 85% of sporadic CRC. Therefore, APC status could
dramatically affect expression and function of PPARγ and the steady-state levels of
PPARγ reported here do not exclude loss of PPARγ transcriptional activity due to somatic
mutations [17], alterations in intracellular distribution [46], post-translation modifications [47] or inhibition by PPARδ [48].

Like PPARγ, PPARδ gene expression is detected in the colon and the receptor can be
activated by fatty acids and derivatives. Herein, we reported an elevated level of
PPARδ (~18%) in CRC. Upregulation of PPARδ gene expression might be attributed to
deregulation in the APC/β-catenin pathway since PPARδ is considered to be a downstream
target gene [20]. Increased levels of PPARδ expression have already been observed in rodent colorectal
tumors and in primary human colorectal adenocarcinomas [20,21]. Nevertheless, PPARδ function remains elusive, with data showing that PPARδ was dispensable
for polyp formation [49]. Our data and others suggested a contribution of PPARδ in the carcinogenesis process
[16,50] while Marin et al. [22] described that agonist-activated PPARδ protects against cancer development. As for
PPARγ, the integrity of the tumor suppressor APC might be essential to guarantee PPARδ
normal function.

Critical to the transcriptional activity of PPARs is the ability to form a complex
with RXR and bind to DNA. Synthetic ligands of RXRα were shown to exhibit insulin-sensitizing
activity [51,52] and to act synergistically with PPARγ ligands to enhance PPARγ/RXRα-mediated transactivation
[31]. In addition, a positive correlation in healthy mucosa was found between RXRα and
PPARγ supporting the idea of a tight relationship in the regulation of the expression
of these receptors. While no change in RXRα expression level was previously noted
in 17 patients with CRC [41], our data revealed a significant increase in tumor versus normal tissue. A similar
upregulation was also observed in human esophageal [53], breast [54] and hepatocellular carcinomas [32]. However, little is known about the function of RXRα in colon tumorigenesis and our
results do not rule out the possibility of alterations in RXRα functions due to altered
localization [55] or inhibitory effect of unliganded RXRα on PPARγ transactivation [56].

Recent data have also suggested that PPARγ anti-tumor activity required a functional
RARβ [57]. This implies that PPARγ function may be affected by alterations in the retinoid
pathway. To our knowledge, very few reports have examined the expression of retinoid
receptors in CRC. Therefore, we described here the first detailed analysis of nuclear
receptor RARα, β and γ mRNA expression in CRC. RARβ has been extensively studied in
cancer cells and human carcinomas, and several studies have suggested that it may
play a role as a tumor suppressor gene [58-60]. However, our results showed a significant upregulation in the expression of all
three RAR isotypes in CRC specimens compared to adjacent normal mucosa. Furthermore,
we showed a complex association between the expression of mRNA for RXRα, RARs, and
PPARs in cancer tissue, suggesting interactions and cross-talk between these receptors
in tumorigenesis. These results demonstrated that alterations are not restricted to
a single receptor. Instead, we observed a profound dysregulation of the retinoid pathway
in this CRC. Downregulation of mRNA expression of RARβ often observed in cancer cells
has been considered as a cellular mechanism to prevent retinoid-induced growth arrest
[61,62]. On the other hand, while elevated levels of RAR mRNA expression has also been described
in breast, liver and esophageal tumors [53,63,64], mechanism and significance are unknown. Nevertheless, if the expression of RAR correlates
with tissue sensitivity to retinoids, our results should be confirmed within a larger
number of samples and both the mechanism leading to inappropriate RXR and RAR expression
and the response of CRC to retinoids should be investigated.

There are strong correlations between the intake of fatty acids, the establishment
of metabolic disorders and an increased risk of developing CRC [65,66]. This suggests the involvement of PPARs and retinoid receptors, activated by fatty
acids and derivatives [21] and modulated by metabolic disorders [67] in establishing a link between overweight prevalence and CRC pathogenesis. In the
current report, we aimed to clarify whether aberrations in the expression of nuclear
receptors may contribute to associate high BMI and CRC. However, alterations in nuclear
receptor expression observed in tumors were similar in both patients with low or high
BMI. We also investigated COX-2 expression which is involved in cellular responses
to lipids and inflammatory processes that favour tumorigenesis by stimulating cell
proliferation and angiogenesis [68]. Interestingly, while COX-2 was greatly expressed in CRC as previously shown [69], we also found a significantly increased level of COX-2 expression in normal mucosa
from patients with high BMI compared to low BMI patients. Recently, it has been reported
that patients with a high risk of developing CRC presented an upregulation of the
COX-2 gene in normal-looking colon mucosa [70]. This supports the idea that COX-2 deregulation might be an early event in the process
of carcinogenesis. Nevertheless, although previous reports showed COX-2 regulation
by nuclear receptors [31,71], very few associations were found in our study between COX-2 and nuclear receptor
expression. In conclusion, our study described altered expression of nuclear receptors
in CRC specimens. Further studies are warranted in order to determine the underlying
mechanism leading to altered expression of PPARs and retinoid-activated receptors
and the significance of such alterations. Moreover, alterations in nuclear receptor
expression were independent of the weight status of patients. Nevertheless, COX-2
might be one early target influenced by excess weight and associated metabolic disorders
and consequently might affect nuclear receptor expression and activation.

Authors' contributions

BD and PC designed the research plan; AR and ER provided and analyzed specimens (histopathology);
BD performed research; MC performed statistical analyses; BD and PC analyzed data;
and BD wrote the paper. All authors read and approved the final manuscript.